Safety assessment of five candidate probiotic lactobacilli using comparative genome analysis

Micro-organisms belonging to the Lactobacillus genus complex are often used for oral consumption and are generally considered safe but can exhibit pathogenicity in rare and specific cases. Therefore, screening and understanding genetic factors that may contribute to pathogenicity can yield valuable insights regarding probiotic safety. Limosilactobacillus mucosae LM1, Lactiplantibacillus plantarum SK151, Lactiplantibacillus plantarum BS25, Limosilactobacillus fermentum SK152 and Lactobacillus johnsonii PF01 are current probiotics of interest; however, their safety profiles have not been explored. The genome sequences of LM1, SK151, SK152 and PF01 were downloaded from the NCBI GenBank, while that of L. plantarum BS25 was newly sequenced. These genomes were then annotated using the Rapid Annotation using Subsystem Technology tool kit pipeline. Subsequently, a command line blast was performed against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) to identify potential virulence factors and antibiotic resistance (AR) genes. Furthermore, ResFinder was used to detect acquired AR genes. The query against the VFDB identified genes that have a role in bacterial survivability, platelet aggregation, surface adhesion, biofilm formation and immunoregulation; and no acquired AR genes were detected using CARD and ResFinder. The study shows that the query strains exhibit genes identical to those present in pathogenic bacteria with the genes matched primarily having roles related to survival and surface adherence. Our results contribute to the overall strategies that can be employed in pre-clinical safety assessments of potential probiotics. Gene mining using whole-genome data, coupled with experimental validation, can be implemented in future probiotic safety assessment strategies.


INTRODUCTION
Micro-organisms from the Lactobacillus genus complex (LGC), occurring naturally or intentionally introduced, are commonly found in fermented food items and are extensively utilized as probiotic dietary supplements [1].These bacteria inhabit the oral cavity, digestive system and female reproductive and urinary tract in humans [1].The majority of lactic acid bacteria are considered suitable for supplement use and oral consumption due to their documented history of safety [2].The European Food Safety Authority has identified a list of species presumed safe for oral consumption under the 'Qualified Presumption of Safety' concept introduced in 2007 [2].This list serves as a justifiable basis for food safety, backed by extensive empirical studies and reviews.Similarly, the procedures for assessing food safety in the United States involve the generally recognized as safe (GRAS) regulation [2].Despite having a list of probiotics considered safe, strain-specific testing must still be employed, as lactobacilli can, in some instances, lead to infections such as endocarditis, bacteremia, neonatal meningitis, dental caries, intraabdominal abscess, pulmonary infections, pyelonephritis and pleuropneumonia [1].Lactobacilli rarely cause disease in immunocompetent individuals but can cause infections when risk factors and underlying conditions are present (e.g.diabetes mellitus, pre-existing structural heart disease, total parenteral nutrition, organ transplantation, HIV infection and steroid use) [1].The species frequently implicated in the pathology include Lacticaseibacillus rhamnosus and Lacticaseibacillus paracasei, both of which harbour potential virulence factors.These factors include the production of enzymes that degrade human glycoproteins and proteins that adhere to extracellular proteins like fibronectin, fibrinogen and collagen.Certain strains can aggregate human platelets, while others can bind fibrinogen.These functions assist Gram-positive pathogens in evading the immune system and fostering platelet aggregation, consequently leading to infections like endocarditis [1].
The potential pathogenesis in patients is multifactorial, including probiotic translocation facilitated by a leaky or damaged intestinal barrier, the existence of virulence factors and harmful metabolites predisposing to opportunistic infections and metabolic disturbances, horizontal gene transfer of antibiotic resistance (AR) genes from probiotics to pathogenic gut bacteria, and an exacerbated immune response elicited through cytokine production [3].Pathogenic bacteria have the capacity to acquire antibiotic resistance genes from probiotics through horizontal gene transfer.In instances where the probiotic displays intrinsic resistance, the implication is that all antibiotic resistance genes are inherent to all strains within that species [4].In contrast, when species traditionally susceptible to a drug exhibit resistance, it is considered as acquired resistance [4].The latter may arise from the acquisition of exogenous DNA or inherent mutations within indigenous genes.Acquired resistance is frequently perceived with a high potential for horizontal spread [4].
Pathogenicity of organisms is characterized by the ability to colonize, invade and damage the host, causing illness [5].Most pathogens utilize a combination of two properties to induce disease: (1) toxicity, representing the degree to which a substance can cause harm, and (2) invasiveness, the ability to penetrate the host and spread [5].The ultimate outcome of the disease process depends on the balance between the microbe's pathogenicity and the host's immune response status [5].In healthy individuals, opportunistic pathogens cannot initiate infections as they lack the necessary mechanisms of toxicity and invasiveness to overcome the immune system [5].In some cases, opportunistic pathogens can cause infections in the setting of a weakened immune response, an altered microbiota or dysbiosis, and invasive procedures/medical devices [5].
The intricate crosstalk between the host and the gut microbiota is essential for maintaining intestinal homeostasis.This balance between different species within the microbial community is termed eubiosis, and any disruption of this balance is referred to as dysbiosis [6].In a setting of eubiosis, or a healthy and balanced microbial ecology the commensal bacteria prevent opportunistic pathogen infection through microbial competition, antimicrobial production, mucosal barrier integrity maintenance and immunomodulation [7,8].The healthy commensal bacteria exert their immunomodulatory functions in a variety of ways.For instance, the gut microbiota plays a crucial role in regulating the expansion of specific lymphocyte subsets, particularly T helper 17 (TH17) cells.TH17 cells are vital for

Impact Statement
Probiotics are micro-organisms that are often incorporated in fermented foods and supplements due to their intended health benefits when consumed orally.While these bacteria are generally considered safe, there is a lack of comprehensive data regarding their potential to cause disease.Bacteria belonging to the Lactobacillus genus complex rarely cause disease in immunocompetent individuals.Some species have been implicated in infections and have been hypothesized to contain virulence factors that enable them to cause infections more effectively in vulnerable groups.Therefore, it is important to understand factors that could cause disease in susceptible populations that may be present in potential probiotics.This study aims to investigate the genetic similarities between selected lactic acid bacteria (LAB) and pathogenic bacteria.Specifically, the study aims to compare the genes associated with virulence found in pathogenic bacteria, through an existing database, with those found in LAB.Through comparison of these genetic similarities, the study aims to gain insights into the role of 'virulence and pathogenic factors' found in disease-causing bacteria that are present in selected LAB.The research can help improve our understanding of the potential risks of probiotics and aid in the development of safer probiotic products for vulnerable populations.
host defence, and the production of pro-inflammatory cytokines, IL-17A, IL-17F and IL-22 [6].Some specific species of commensal bacteria have been reported to induce the generation of regulatory T (Treg cells), recognized for their regulatory role in the immune system, through TGF-beta activation in epithelial cells.Consequently, disruptions in the healthy microbiota often result in issues with immunomodulation and local defenses on the intestinal epithelium, as observed in autoimmune diseases and the increased susceptibility to opportunistic pathogens [6].For instance, as a consequence of antibiotic use, commensal micro-organisms are often killed in the process.This results in a reduction of micro-organism-mediated innate immune defenses, allowing residual antibioticresistant opportunistic pathogens to inhabit the mucosal surface, such as cases of C. difficile infections [8].
The ability of the host to discriminate between pathogenic and commensal bacteria is still poorly understood.It is believed that the sequestration of indigenous microflora by surface epithelium inhibits TLR activation by commensal bacteria [8].In contrast, pathogenic bacteria possess virulence factors that aid them in traversing the epithelial barrier, allowing recognition by TLRs expressed on dendritic cells and macrophages [8].Conventional virulence factors include protein toxins and enzymes, cell-surface structures, capsular polysaccharides, lipopolysaccharides and outer membrane proteins, with each playing a direct role in the progression of the disease [9].The general concept of bacterial pathogenesis is dependent upon host susceptibility and bacterial infectivity [10].Virulence factors enable bacteria to successfully invade the host, induce disease and circumvent host defenses [10].These factors can be categorized as adherence factors, invasion factors, capsules, exotoxins, endotoxins and siderophores [10].Bacterial invasion happens because of complex interactions between a prokaryotic cell and the host target cell.The general steps happen as follows: (1) adherence to eukaryotic cells, (2) entry of the bacteria into the body, (3) avoidance of host immune mechanism, (4) tissue damage and functional impairment and colonization of host tissues and (5) resistance to antibacterial agents [10].The presence of virulence factors within the genome, however, does not inherently predict their potential to be virulent or cause disease.In some cases, a mutation in a virulence factor from a pathogenic strain may attenuate the pathogenicity.Virulence factors may also be present in attenuated and even avirulent strains [11].
Understanding the mechanisms that enable pathogens to cause diseases is key in treating pathogenic infections and may predict the potential dangers of candidate probiotic strains.Candidate strains are often evaluated for their safety by assessing resistance to antibiotics, and checking for the presence of antibiotic resistance genes, and virulence genes [12].Additionally, the haemolytic activity, as well as the bile salt hydrolase activity, is often determined at the strain level; however, their importance in probiotic safety is still unclear [2].In this study, five candidate probiotic strains, Limosilactobacillus mucosae LM1 (CP011013.1),Lactiplantibacillus plantarum SK 151 (CP030105.1),Lactiplantibacillus plantarum BS25 (JAVBIR000000000), Limosilactobacillus fermentum SK 152 (CP016803.1)and Lactobacillus johnsonii PF01 (CP024781.1)were screened against a database of genes that play a role in infections and host colonization in pathogenic bacteria.These strains were also subjected to antimicrobial resistance (AR) screening and identification.These strains were chosen because of their notable probiotic potential [3,[13][14][15][16][17][18][19][20][21].To date, there is no extensive study exploring the potential pathogenicity of the five strains.It is important to screen for potential genetic factors that cause disease in a human host.

Genome and annotations
The RAST annotation pipeline was used to annotate and view the genomic data of all strains.The genome sequences of LM1, SK151, SK152 and PF01 were downloaded directly from the NCBI GenBank, while that of BS25 was sequenced by the Philippine Genome Center [13].Genome annotations ran through the Rapid Annotation using Subsystem Technology tool kit (RASTtk) version 1.3.0pipeline with the following default settings: preserve gene calls disabled, automatically fix errors enabled, fix frameshifts disabled and backfill gaps enabled [22].

Virulence factor gene mining
A local command line blast search was conducted using blast +software version 2.2.30+.The genome sequences of LM1, SK151, BS25, SK152 and PF01, previously annotated, were utilized, and blast searches were conducted against the Virulence Factor Database (VFDB) containing protein sequences from a core dataset that includes experimentally verified virulence factor genes [23].The following parameters of blast were applied, with an e-value threshold of 1e-5, max thread of 4, and a maximum target sequence set to 1. Subsequently, the blast outputs were filtered for hits that met a minimum identity of 70 %.In addition, the SEED viewer in the RAST annotation pipeline lists all possible virulence factors according to their database and these were tabulated.

Antibiotic resistance gene mining
The annotated genes of the strains were queried against the Comprehensive Antibiotic Resistance Database (CARD) using the Resistance Gene Identifier (RGI) web portal version RGI 6.0.3,CARD 3.2.8 using the default parameters: perfect and strict hits only, exclude nudge, and high-quality coverage, to identify antibiotic resistance (AR) genes that exhibit potential for horizontal gene transfer [24].Additionally, the five strains were also run through ResFinder 4.0 to determine acquired AR genes using default parameters and a 90 % ID and 60 % minimum length threshold [25].

Genome visualization and comparisons
Visualization of the genomes and annotated genes were visualized and compared using Artemis and the Artemis comparison tool version 18.2.0,and the SEED viewer linked with the RASTtk (version 1.3.0)annotation pipeline [22,26].

RESULTS AND DISCUSSION
Gene annotation reveals genes associated with virulence, disease and defense Gene annotation was performed using the RASTtk (version 1.3.0)pipeline, and genes associated with virulence, disease and defence were identified and visualized using the SEED viewer [22].A total of 47, 48, 48, 37 and 48 associated genes were categorized for LM1, SK151, BS25, SK152 and PF01, respectively.Figs 1-5 show the gene map of the five strains and the grouping of the annotated genes into specific categories.The predicted genes were categorized using the SEED viewer (Table 1).The majority of the genes were categorized under resistance to antibiotics and toxic compounds.Notably, SK151 contains multiple genes related to beta-lactamase resistance, which may raise safety concerns particularly in the possibility of horizontal gene transfer to pathogenic bacteria.
Bile salt hydrolase (BSH) encoding genes were identified in LM1, SK151, BS25 and PF01 strains with one, three, three and five homologues, respectively.The presence of BSH is crucial for the survival of probiotics, as host bile acids can act as bacteriostatic agents, negatively impacting intestinal flora by dissolving bacterial membranes [27].This enzyme can detoxify bile, facilitating the colonization of lactic acid bacteria in the gastrointestinal tract [28].Many probiotic strains harbour multiple BSH homologues, providing a potential survival advantage.Each homologue may exhibit distinct responses to various bile types or exposure durations, ultimately maximizing survival [28].Currently, there is no literature implicating any potential for pathogenicity associated with the presence of BSH encoding genes, but its importance may be hypothesized to play a role in documented cases of cholecystitis associated with lactic acid bacteria [29].In addition, no virulence factors involved in human host damage, haemolysis  and toxin production were detected.The possible factors that could play a role in pathogenicity (e.g.adhesion mechanism and bile salt hydrolase activity) contribute to the survival of micro-organisms in the mammalian gut and such are characteristics of much of the local microbiota.It is therefore not recommended to use these factors as a basis for pathogenicity as they are not a priori meaningful measures of virulence [30].

Presence of survival and virulence factors and their implications on the safety of probiotic candidates
LM1, SK151, BS25, S152 and PF01 were queried against a database downloaded from the Virulence Factor Database (VFDB; http://www.mgc.ac.cn/VFs) core dataset, and command line blast was performed using the annotated sequences [23].The VFDB core dataset is composed of virulence factors experimentally verified across the literature [23].A total of 4, 8, 6, 1, and 2 hit/s (excluding repeat hits) were obtained from LM1, SK151, BS25, SK152 and PF01, respectively.The listed hits from the database in Table 2 were tabulated based on percent ID and bit score, omitting repeating genes.All five strains identified the eno gene.The L. plantarum strains SK151 and BS25 share matches with the following genes: eno, clfA, sdrE, sdrF and sdrG.The genes identified are characterized by functions related to surface adhesion, immunomodulation and platelet aggregation, and each will be discussed in further detail.No genes were detected related to haemolysis using the RAST annotation as well as the local blast with the VFDB core dataset.
Table 2 Top sequence matches of the queried strains against the VFDB with their function and potential role in virulence.
It has been mentioned previously that the presence of haemolytic genes must be assessed in candidate probiotic strains to evaluate their safety.The presence of haemolytic activity in probiotics is undesirable as it can cause anaemia and oedema in the host.It is important to screen for haemolysin produced by a strain as it breaks down host cells, releasing iron-containing compounds like haemoglobin [31].If the strain exhibits β-hemolytic activity, it is considered harmful, whereas γ-haemolytic and α-haemolytic activities are generally considered safe [31].The blast results against the VFDB for the five candidate strains showed no genes related to haemolysis but in vitro testing is still recommended.
Immunomodulation and surface adhesion are some of the key functions of lactic acid bacteria to establish initial gut colonization.The results from the blast reveal an identical gene, tufa, present in lactic acid bacteria strains BS25 and PF01, analogous Involved in biofilm formation [42] Table 2. Continued to that of pathogenic bacteria Tularensis subsp.holarctica and Mycoplasma mycoides subsp.mycoides, respectively.The tufa gene encodes an elongation factor Tu (Ef-Tu) G protein, which facilitates the binding of aminoacyl-tRNA to the A-site of the ribosome within living cells [32].In pathogenic Gram-positive and Gram-negative bacteria, Ef-Tu showcases a range of diverse roles.These functions include binding to immune system regulators, enhancing virulence, promoting immune evasion, facilitating invasion of host cells, and interacting with Mreb to regulate cell shape.In addition, Ef-Tu can be fragmented via proteolytic processing, acting as molecular decoys that, in turn, promote immune evasion [32].In contrast, in some Lactobacillus species, Ef-Tu performs various functions, such as attachment to human intestinal cells, host immunomodulation, mucin binding and actin bindin [33].
A study by Granato et al.Granato et al. (2004) on Lactobacillus johnsonii reported that Ef-Tu, in this species, binds to human intestinal cells and mucins in a pH-dependent manner, suggesting its contribution to gut colonization [34].Additionally, this function enables lactic acid bacteria to exert beneficial effects by competitively adhering to the intestinal mucosa, thereby excluding potentially pathogenic bacteria and preventing infections [34].The protein RfbB from the reference strain Streptococcus gordonii str.Challis, was matched with a protein found in the LM1 strain.RfbB, also known as dTDP-glucose 4,6 dehydratase, is involved in the biosynthesis of dTDP-rhamnose and immunoregulation.It has been shown to be upregulated in the setting of an attenuated immune response of macrophages [35].In Salmonella typhimurium, it has been reported that the function of RfbB is to catalyse the synthesis of both O-antigen and the enterobacterial common antigen [36].These lipopolysaccharides play a crucial role in maintaining the structural integrity of the outer membranes of Gram-negative bacteria.The functional loss of RfbB, using a gene deleted mutant of S. typhimurium, resulted in defects in outer cell-wall permeability, leading to hypersensitivity to bile and cell-wall-targeting antibiotics.Additionally, the loss of the protein led to a reduced production of pro-inflammatory cytokines [36].This highlights the potential role of RfbB in cell-wall integrity, immunomodulation and virulence in S. typhimurium [36], and it can be hypothesized to function similarly in the probiotic strain LM1.
The strains BS25, PF01, SK152 and LM1 have hits with various Streptococcus species on the eno gene, which codes for the protein phosphopyruvate hydratase belonging to the enolase family.Enolase is a metalloenzyme that catalyses the reversible conversion of 2-phospho-d-glycerate (2-PGE) to phosphoenolpyruvate, a pivotal reaction in glycolytic pathways across diverse organisms, including bacteria.Surface-associated enolase has been observed to interact with the human host in streptococcal infection cases.
In the pathogenesis of S. pneumoniae and S. pyogenes, it engages with human plasminogen (Plg), enhancing proteolytic plasmin activity, which plays a pivotal role in the progression of pneumococcal and group A streptococcal infections [37].In contrast, a gene found in the study by Castaldo et al.Castaldo et al. (2009) revealed an enoA1 gene, coding for EnoA1 alpha-enolase, which plays a role in fibronectin binding [38].Fibronectin is present in the extracellular matrix of intestinal epithelial cells, and surface adhesion through fibronectin binding is essential for probiotic transit and potential integration with the intestinal mucosa [38].Currently, no literature can be found regarding the role of enolase in BS25, PF01, SK152 and LM1 strains concerning Plg interactions.It has been established that efficient probiotic Lactobacillus strains share certain characteristics with pathogens, including survival and adherence factors crucial for competition with pathogens.Existing data also suggests that probiotic lactobacilli interact with immune cell receptors and influence the functions of epithelial cells.Some of these interactions parallel those observed in pathogens but are not present in commensal and resident bacteria.While some pathogenic bacteria use certain mechanisms to evade the immune system or downregulate the immune response, lactobacilli utilize similar mechanisms but instead produce immune response-promoting effects [39].
The aggregation of blood platelets by bacteria is believed to contribute to the pathogenesis of infective endocarditis, which results from the deposition of platelet fibrin clots on the endothelial surface of the heart [40].Platelet aggregation has been more extensively studied and reported in Staphylococcus aureus, Streptococcus sanguis and group B streptococci but studies on Lactobacilli are limited.A study by Harty et al.Harty et al. (1993) reported that certain lactobacilli can aggregate human platelets with particularly increased frequency in L. rhamnosus strains [40].The gene GspB, producing a cell-wall-anchored glycoprotein, was identified in strain LM1.This gene encodes a cell-surface protein crucial for platelet binding in Streptococcus gordonii strain M99 [41].GspB shares identity with a protein called Hsa, expressed by S. gordononii Challis [41].GspB facilitates the binding of various carbohydrates containing sialic acid in either α(2-3) or α(2-6) linkages [41].A notable difference between GspB and Hsa lies in their localization: despite its significant role in adherence to sialic acid, most of the Hsa expressed in S. gordononii Challis remains within the cytoplasm [41].Hsa is also recognized as a sialic-binding haemagglutinin and has been demonstrated to have roles in platelet agglutination [41,42].The potential of LM1 to induce blood platelet aggregation and cause infective endocarditis is still uncertain and requires further testing.
The strains SK151 and BS25 both belong to the Lactiplantibacillus planatrum strain, and have been shown to have genes related to Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) identical to those found in Staphylococcus species.S. aureus has been known to have these MSCRAMMs, which allow it to survive in a variety of tissue cells [43].MSCRAMMs are adhesins characterized by the presence of a minimum of two IgG-like folds and utilize a ligand-binding mechanism known as the 'close, dock, lock and latch (CDLL)' mechanism.The MSCRAMMs recognize and bind to extracellular matrix proteins, initiating the pathogenic process.Typically, MSCRAMMs are known to interact with fibrinogen, fibronectin, neurexin and IgGs.Staphylococcal MSCRAMMs, belonging to the Clf-Sdr protein family, include clumping factor A (ClfA), clumping factor B (ClfB) and the Sdr proteins, such as SdrC, SdrD and SdrE in S. aureus, and SdrF and SdrG in S. epidermidis.
In S. aureus, both ClfA and ClfB serve as fibrinogen-binding proteins and are upregulated during biofilm growth.ClfA induces bacterial clumping when combined with fibrinogen [44].Additionally, SdrC facilitates bacterial intercellular interactions and contributes to biofilm formation.Even in the non-pathogenic bacterium Lactococcus lactis, there is evidence suggesting that SdrC promotes bacterial adhesion to abiotic surfaces.Additionally, studies suggest that the involvement of SdrC in biofilm formation varies based on the strain background [45].SdrD plays crucial roles in both colonization and infection by promoting bacterial survival in blood and inhibiting human neutrophil activity.In addition, it inhibits innate immune-mediated bacterial killing independently of other S. aureus proteins.In the case of non-pathogenic bacteria, introducing recombinant SdrD and expressing it heterologously in Lactococcus lactis have both been found to enhance its survival in human blood [43].The protein SdrD has been shown to exhibit an immune evasion tactic in S. aureus.When it binds to human complement factor H (CFH) via the CDLL mechanism, it captures the C-terminal tail of CFH, and the process sequesters CFH on the bacterial surface, resulting in complement evasion [46].Some strains of S. epidermidis can attach to fibrinogen immobilized in a plastic surface, which increases the likelihood of device-related infections when the device is implanted in the human body, and it has been shown that the protein SdrG plays a significant role in this process through adherence to fibrinogen.Regarding its role in lactic acid bacteria, studies have shown that a non-pathogenic strain Lactococcus lactis MG1363 made to express high levels of SdrG through an expression vector showed that these strains strongly adhered to immobilized fibrinogen [47].Lastly, the SdrH protein is implicated in the initial attachment of the bacterium during biofilm formation on a surface.It was observed to be upregulated during the initial 4 h of biofilm formation in S. epidermidis, although the exact mechanisms for this process are yet to be elucidated [48].Based on the presence of Sdr encoding genes in L. plantarum species it could be hypothesized that they are capable of biofilm formation as well as an enhanced tendency to survive in the bloodstream.It is also important to note that both L. plantarum BS25 and SK151 share multiple Sdr proteins found in both S. aureus and S. epidermidis, indicating their propensity for possible biofilm formation and device-related adhesion.However, there is not enough evidence to suggest the likelihood of bloodstream infections associated with L. plantarum especially in immunocompetent individuals.It was noted above that engineered Lactococcus lactis with elevated expressions of sdrC and sdrD had improved surface adhesion to abiotic surfaces and improved survival in blood [43,45], respectively, but these alterations do not necessarily entail any increased risk of bacteremia.One explanation for the low pathogenicity and virulence of lactobacilli in healthy human hosts can be attributed to their lack of tissue destruction and 'true' virulence factors.Consequently, their interaction with the host is generally favourable [39].The majority of the genes presented above have roles in biofilm formation, surface aggregation, immune detection and immunomodulation and have not yet been fully explored in all five strains.To date, no literature thoroughly explores the role and regulation of these genes in pathogenicity for lactic acid bacteria.
Although multiple hits were derived from the VFDB as presented above, it does not predict the pathogenicity of the five LAB strains.The common functions of the genes recognized through the VFDB are related to survival, surface adhesion and recognition of the host immune response.As mentioned previously, the mechanisms of pathogenicity of Lactobacillus spp.are often through the breakdown of human glycoproteins, proteins that bind extracellular proteins, interactions with fibrinogen, and the ability to aggregate platelets.Genes identified using the VFDB are not implicated in the observed mechanisms of lactic acid bacteria pathology and are mostly related to survival and surface adhesion.Land et al.Land et al. (2005) reported two patients who were administered probiotic bacilli later experienced bacteremia and sepsis linked to Lactobacillus species [49].Subsequent molecular fingerprinting indicated that the Lactobacillus strain identified in the blood sample was identical to the probiotic strain consumed.This case then concludes that even evaluated and approved Lactobacillus strains have the propensity to cause disease in certain populations.It is important to mention that the cases mentioned previously are paediatric cases that have concomitant diseases (i.e.heart valve disease and UTI).Therefore, it may be a question of host susceptibility rather than the presence of acquired survival factors of Lactobacillus that cause disease.This does not however discourage the use of probiotics but rather caution its use on certain populations [49].Assessing the potential virulence factors in bacterial groups without recognized and documented pathogenic members remains a challenge [49].
In this study, all virulence factors were compared against known virulence factors found in recognized pathogenic bacteria (e.g. S. aureus).No definitive pathogenicity-related genes have been identified in Lactobacillus or Bifidobacterium species used as probiotics [50].Cases of sepsis in vulnerable populations linked to lactobacilli lack conclusive evidence from clinical isolates, suggesting no species-specific properties that can lead to infection [50].In addition, the current obstacle in predicting the pathogenicity of the identified survival and defence factors lies in the fact that these genes have not been thoroughly studied in the context of probiotic infections.Identifying variants of these genes and how they are regulated in members of the LGC that cause infections and bacteremia may yield valuable insights into the process of screening for the safety of potential probiotics.The possibility of enhancing, silencing and selectively regulating these genes for safety remains to be explored.

Antibiotic resistance (AR) genes and horizontal gene transfer (HGT)
One of the potential dangers of introducing a foreign probiotic is the possible integration of antibiotic resistance through horizontal gene transfer toward more pathogenic bacteria.Allard et al.Allard et al. (2002) conducted a study to assess the antibiotic susceptibility of more than 182 Lactobacillus type strains and compared these findings with genome-wide annotations of antibiotic resistance genes (AR genes) [51].They discovered that most had genes encoding resistance to aminoglycosides, tetracycline, erythromycin, clindamycin and chloramphenicol [51].According to the European Food Safety Authority (EFSA), the possibility of transfer of resistance genes to pathogenic bacteria is related to the genetic basis of resistance.The likelihood for intrinsic resistance to undergo horizontal spread is believed to be low [4].
The five strains were also analysed using the Resistance Gene Identifier tool found in Comprehensive Antibiotic Resistance Database (CARD).In addition, the genomes were also analysed using an online bioinformatics tool, ResFinder 4.0, for predictions of phenotypes from AR genes [25].
Further observation of the genomes of the five strains revealed resistance genes to tetracycline (tetW) found on both strains LM1 and PF01.The strain PF01 also had a match to an AR gene related to erythromycin (ermT) on strain PF01.These findings are not uncommon for probiotics.A study by Campedelli et al.Campedelli et al. (2019) showed that many lactobacilli are antibioticresistant [52].Most Lactobacillus strains are intrinsically resistant to aminoglycosides, ciprofloxacin and trimethoprim [52].
Additionally, other reports suggest that some lactic acid bacteria have intrinsic resistance to bacitracin, kanamycin, teicoplanin, vancomycin and beta-lactams [30].Analysis of strains LM1, BS25, SK151 and SK152 revealed glycopeptide resistance gene clusters: vanT gene in vanG cluster, vanY gene in vanB cluster, vanY gene in vanB cluster and vanT gene in vanG cluster, respectively.Strains from fermented foods have been shown to contain acquired resistances to erythromycin, tetracycline, chloramphenicol and clindamycin [52].Studies have also revealed that more than 75 % of antibiotic resistance genes present in the human flora of screened individuals are specific to tetracycline, macrolides and beta-lactams [30].Results from ResFinder 4.0 confirmed the presence of tetracycline and erythromycin resistance genes in PF01.In addition, ermT and tetW hits were also identified as acquired AMR gene hits in PF01.Similarly, tetW was identified as an acquired AMR gene in LM1.No acquired AMR gene hits were identified for BS25, SK151 and SK152.In the context of possible horizontal gene transfer to pathogenic bacteria, it is important to consider that most horizontal transfer events in the bacterial chromosome are often deleterious and there is a low mechanistic probability of HGT occurrence and may take months to years to occur [53].

CONCLUSION AND FUTURE PERSPECTIVES
The study gathered information on genes found in selected probiotic strains that may cause harm in humans using RAST gene annotation, VFDB, AR gene mining (ResFinder), and manual identification through the related literature.The majority of the genes categorized under 'virulence, disease and defence' identified using the RAST pipeline are associated with bacterial resistance to drugs and environmental conditions.This study is still limited by the fact that the virulence factors in the database are based on known pathogenic bacteria and may exhibit different mechanisms of pathogenicity.To date, there is no known 'true' pathogenic LAB, which serves as an obstacle in predicting virulence factors specific to LAB and its respective species.There is no absolute pathogenic strain representative of the five species/strains that can help narrow down significant virulence factors for these potential probiotics.The genes mined using the VFDB mostly reported genes associated with biofilm formation, surface adherence, platelet aggregation, survival and host immune modulation and would be misleading to be considered as virulence factors in the context of probiotics.No genes were detected associated with toxins and superantigens.All five strains contain potential intrinsic antibiotic resistance genes related to tetracycline and erythromycin.The potential pathogenicity of the five LAB strains is possibly dependent on the host immune response.It is important to note that even probiotic strains approved and commercialized have been known to cause bacteremia in susceptible individuals, but no universal genetic markers have been identified that can predict their pathogenicity.Mutational analysis of the mined survival factors with a special attention to fibrinogen-binding genes should be performed, as they are commonly associated with a disease process in isolated cases.It would also be beneficial to have a database of cases with lactic acid bacteremia to create a comprehensive database of gene variants that can cause pathology.
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Your submissions to our titles support the community -ensuring that we continue to provide events, grants and professional development for microbiologists at all career stages.Comments: Overall the manuscript has improved, however there is still a number of things that need to be adressed: 1.5 Repositories -add the name of the repository here as well not just accession number Reference 3 is not an actual referenceplease correct this and add an appropiate reference-this looks like some automatically generated suggestions l. 30 pathogenesis is wrongly used here, I think you mean pathogenecity since the bacteria do not develop a disease but rather cause it Abstract is missing a final summary sentence of what your findings contribute or how they can be used for policies, what should happen next?l. 76 Lactobacillus should be in italics l. 83 please add the reference to this documentation?l. 85-86 Reference Binda et al 2020 is in a different format than the rest of the references l 96-97 "strains have the ability to" replace by "strains can" l 101 "of disease" is redundant when using pathogenesis is used prior l 172-175 species names need to be in italics l 180 change " may cause a disease process" to "cause disease" L 185 RAST version needs to be stated L193 be more specific which sequences are you referring to here?It is assumed the 5 genomes?! Ll 209-211 references for the tools are missing L 225 would add "encoding" before genes, bsh should be in capital letter as you are referring to an enzyme L 226 bsh should be Bsh as I presume you are referring to the enzyme and not the gene L 230 suddenly it is BSH here, it should be consistent throughout.Genes need to be small letters and in italics, proteins first letter in capital and no italics L233 bsh genes -this needs to be in italics L 239 I think you mean here "a priori Please find below our response to the reviewers' comments on our manuscript, with the revised title, "Safety Assessment of Five Candidate Probiotic Lactobacilli using Comparative Genome Analysis, " with manuscript number ACMI-D-23-00183.The revisions in the manuscript file are highlighted in yellow.
We thank you and the reviewers for your thoughtful suggestions and insights.We hope that our response will merit your favorable approval on allowing our paper to be published in your prestigious journal. Sincerely,

EDITOR COMMENTS RESPONSE
Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included.
Methodology and software versions have been updated.
For the purpose of clarity I suggest you put a line which repositry you have used under the data statement.
Under data summary please also mention the accession number of the strain you have sequenced as part of your study.
We have added the repository and accession numbers of all strains in the data statement.
It would help clarity if you introduced bacteriocins in the introduction as refer later to why analysing this is of importance and include this in your discussion as well.
We have removed the section and all mentions of bacteriocins in the manuscript, as it does not contribute to the objectives and narrative of our study.
Please state the version and settings for each pipeline that was used (missing for RAST and also under 6.5) We have added the settings enabled and disabled for RAST.
The results appear very much as a list of different findings (and there seems to be a high similarity phrases from references used-you need to address this), I highly recommend to connect/relate these findings in the text and establish more of a narrative for the reader (please also see point 4 from reviewer 1) We have paraphrased the sentences in the author's words.A more narrative form has been established connecting the probiotic strain matches with that of the described genes.
I also strongly agree with the suggestion to rephrase your title.This would also aid establishment of a narrative.
We have rephrased the title to establish our narrative and provide a more concise representation of our study.

COMMENTS REVIEWER 1 RESPONSE
I think the current title of the manuscript should be reconsidered.Aspects of the title could be more concise, i.e. is it necessary to list each candidate probiotic?Furthermore, the element of investigating genetic similarities between pathogenic bacteria and LAB should be more clearly reflected in the manuscript title.
We have rephrased the title to focus on probiotic safety.
In the Results and Discussion, I suggest that sections 7.2 and 7.5 are combined.Currently, section 7.2 focuses on discussing the identified virulence genes, but in the context of pathogens.I would argue that these should be discussed in relation to the candidate probiotic strains / lactic acid bacteria, which is somewhat done in section 7.5.By integrating sections 7.2 and 7.5, the main focus of the manuscript (i.e.probiotics/LAB) would be maintained, and the narrative easier to follow.
We have merged Sections 7.2 and 7.5, and added discussions on the possible role of the detected genes with regards to the candidate LAB.
It is not clear what the bacteriocin mining aspect adds to the manuscript.Furthermore, in section 7.3, there is no discussion of the bacteriocin mining results.I suggest that the authors should reconsider the purpose of bacteriocin mining in this manuscript, or provide justification for its inclusion in the manuscript.
The section on bacteriocin mining has been removed.The order of the references has been fixed.
Line 173: Provide some appraisal of the potential implications of this statement i.e. multiple genes related to beta-lactamase resistance We have added a line stating the potential risk of horizontal gene transfer.
Line 175-176: Provide a citation for this statement We have added a citation on this statement.
Line 300: Correct spelling should be "Campedelli" We have corrected the spelling.
Details of Reference number [7] are incomplete/ incorrect The reference manager generated an incorrect reference but this has been corrected.

COMMENTS REVIEWER 2 RESPONSE
The MS has 38% similarity to references that is not acceptable.Please make sure you use your own words.
We have paraphrased the manuscript text to avoid a high similarity with the references.
The article deals with comparative genomics of Lactobacillus The author failed to incorporate reference for "Pro-biotics….Gras status".
We have added the appropriate reference for this statement.
The safety and toxicity investigations of probiotic microorganisms are the main area of interest for the author.Its hemolytic nature is one of the primary determinants of safety and toxicity.Although the author makes no mention of it, bioinformatics technologies may also be used to anticipate this.
We have added a paragraph on hemolysis.The VFDB core dataset also accounts for hemolysin.
Bile salt hydrolases, the adhesion genes, must be taken into account for the investigation.The author should demarcate survival and adaptation factors of the control, and various microbes considered for the study.Subsequently, they may discuss in comparison with the control so that the study will be much clearer.A small write-up of these factors and their link in the said functions may enhance the credibility.The table may give good clarity.At present the MS looks just WGS analysis data has been exhibited it does not give clear picture of the objectives.
The discussion on bshand adhesions genes have been expounded.The link between the genes matched and the respective probiotics has also been incorporated in the narrative.
The following hypothesis / report needs a reference "Pathogenic bacteria may acquire antibiotic resistance genes from the probiotics via horizontal gene transfer.When the probiotic has an intrinsic resistance, it means that all AR genes are typical of all strains of that species" The citation was clarified.
"In healthy individuals, opportunistic pathogens are not able to produce infections as they lack the necessary mechanisms of toxicity and invasiveness to overcome the immune system".Here the author may need to discuss dysbiosis and eubiosis phenomenon, followed by immune response.
We have added information on dysbiosis and eubiosis.

VERSION 1
Editor Comments: The reviewers have highlighted major concerns with the work presented.Please ensure that you address their comments.Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included.In addition to the points raised by the reviewers, please also consider the following For the purpose of clarity I suggest you put a line which repositry you have used under the data statement.Under data summary please also mention the accession number of the strain you have sequenced as part of your study.It would help clarity if you introduced bacteriocins in the introduction as refer later to why analysing this is of importance and include this in your discussion as well.Please state the version and settings for each pipeline that was used (missing for RAST and also under 6.5)The results appear very much as a list of different findings (and there seems to be a high similarity phrases from references used-you need to address this), I highly recommend to connect/relate these findings in the text and establish more of a narrative for the reader (please also see point 4 from reviewer 1) I also strongly agree with the suggestion to rephrase your title.This would also aid establishment of a narrative.Comments: Please address areas of high similarity concern highlighted on the iThenticate report.The article deals with comparative genomics of Lactobacillus The author failed to incorporate reference for "Probiotics….Gras status".The safety and toxicity investigations of probiotic microorganisms are the main area of interest for the author.Its hemolytic nature is one of the primary determinants of safety and toxicity.Although the author makes no mention of it, bioinformatics technologies may also be used to anticipate this.Bile salt hydrolases, the adhesion genes, must be taken into account for the investigation.The author should demarcate survival and adaptation factors of the control, and various microbes considered for the study.Subsequently, they may discuss in comparison with the control so that the study will be much clearer.A small write-up of these factors and their link in the said functions may enhance the credibility.The table may give good clarity.At present the MS looks just WGS analysis data has been exhibited it does not give clear picture of the objectives.The following hypothesis / report needs a reference "Pathogenic bacteria may acquire antibiotic resistance genes from the probiotics via horizontal gene transfer.When the probiotic has an intrinsic resistance, it means that all AR genes are typical of all strains of that species" "In healthy individuals, opportunistic pathogens are not able to produce infections as they lack the necessary mechanisms of toxicity and invasiveness to overcome the immune system".Here the author may need to discuss dysbiosis and eubiosis phenomenon, followed by immune response.

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data?Partially support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes

Anonymous.
Date report received: 22 October 2023

Line 31 -
32: I think this should state "used for oral consumption" Line has been updated.(Updated Line 29) Line 32: I think this should read "but can exhibit pathogenesis" Line has been updated.(Line 30) Line 39: Please check the phrasing/grammar Line has been updated.(Line 35-41) Line 77: I think this should read "in some instances" Line has been updated.(Line 86) Line 107: check spelling of "status" line has been updated.Line 134: The references cited should be listed in ascending order

Reviewer 1
recommendation and comments https://doi.org/10.1099/acmi.0.000715.v1.3 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Table 2 .
Top sequence matches of the queried strains against VFDB with their function and potential role in virulence It is the ligand for the integrin alphaIIb/ beta3 on the surface of platelets.This binding of fibrinogen to the integrin receptor on activated platelets results in platelet aggregation and the formation of plateletfibrin thrombi.ClfA exhibits fibrinogenbinding characteristics like those of the platelet integrin alphaIIb/beta3.

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*Those in parentheses are the new line/position after changes " L 252 -253 give a bit more detail on the genes here, what do they encode and how are they involved in pathogenecity?Or refer that each will be in detail discussed next.L 267 in vitro in italics, testing not in italics L 281 reference in different format (Granato et al) -no number reference at end of sentence?L 290/294/297 species name should be small letter (T -> t) L 306 species names in italics L 308/309 reference not in number at end of sentence L333-335 please put a reference for this statement L 335 "Hsa expressed in Challis" Challis is a subspecies?Please use the full term.L 382 genes not in italics?L395 pathology should this not rather be pathogenecity?L 428 reference Allard et al 2002 is not at the end of the sentence in number format L429 italics Lactobacillus L 444 Campedelli reference is not at the end of the sentence in number reference format l 445 italics Lactobacillus l 456-7 gene name spelling incorrect Table1Mycobacterium needs to be in italics Table2genes need to be in italics Editor Review Questions This is an open-access article report distributed under the terms of the Creative Commons License.This is an open-access article report distributed under the terms of the Creative Commons License.

recommendation and comments https
://doi.org/10.1099/acmi.0.000715.v1.5 © 2023 Schniete J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.